RL from WGA, and Control Bead Question

[Dynamac]

We recently had six whole genome amplified emPCR's fail. These were from environmental samples. I'm wondering if contaminants have carried through the rapid library prep and killed the reaction. Anybody have similar problems?

Also, what does it mean if control beads are having 60% of their sequences trimmed by the too-short-quality filter? Seems high to me. Only one set of the two control bead types are doing this.

Thanks.

Barry

[bradfish]

Hey Barry,

I have also noticed that one of the control bead types gets lost to short quality a lot more than the other type. No solution, just confirming your suspicions...

[Anthony.287]

Are they the AVF control beads? Those are designed to test the system, and are full of homopolymer regions. You can probably expect a lot of those to fail.
And I've been told that WGA samples are not compatible with the 454. I have more info on both of these subjects; I'll try to check tonight and report back.

[Anthony.287]

Ok, found my notes. They say that WGA is not recommended for the 454 because of artifacts that can be created during the process. Apparently, the polymerase can hop on and off of the DNA, but continue making copies. This leads to chimeras.
As for the control beads, I had a similar, confusing problem. In regions containing a particular customer's samples, on three different occasions, there seemed to be very few, if any, control beads. At first I thought user error. Then it happened again with a different user, then again with yet a different user. It turned out to be the sample. They were relatively short amplicons (~200bp) and amplified very well, so well that the signal from the library was drowning out the controls! I haven't had a chance to resequence these, but our FAS recommended going down to 12.5% of the amplification primer, instead of just 25%.

[Dynamac]

@Bradfish. Thanks for the information.

@Anthony: Well, in this case, the WGA emPCR completely failed. Or perhaps the ligation failed, although I was able to measure incorporated FAM (presumably incorporated and not free FAM). So, I don't think stutter artifacts would kill the emPCR, would they? As for the control beads, it was indeed the AVF set that had the failures. I'll suggest cutting the amount of primer down to the PI. This forum is the first time I've heard of doing that!